Removal of impurities from protein A eluates

ABSTRACT

Modified Protein A, Protein G, Protein L, or Protein A/G that lacks antibody binding activity, and methods of the modified protein&#39;s use for purifying antibodies is provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a divisional of U.S. application Ser. No.14/589,633, filed Jan. 5, 2015, which claims benefit of priority to U.S.Provisional Application No. 61/923,380, filed Jan. 3, 2014, which isincorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Protein A is a 41 kD cell wall protein from Staphylococcus aureas thatbinds with a high affinity to the Fc region of antibodies. For example,Protein A binds with high affinity to human IgG1, IgG2 and IgG4 as wellas to mouse IgG2a and IgG2b. Protein A binds with moderate affinity tohuman IgM, IgA and IgE as well as to mouse IgG3 and IgG1.

Protein G is an immunoglobulin-binding protein expressed in group C andG Streptococcal bacteria. Protein G binds to a broader range of IgGsubclasses than protein A. Protein L was first isolated from the surfaceof bacterial species Peptostreptococcus magnus and also bindsimmunoglobulins via interaction with the kappa light chain.

Protein A/G is a recombinant fusion protein that combines IgG bindingdomains of both Protein A and Protein G. Protein A/G contains four Fcbinding domains from Protein A and two from Protein G.

Each of the above-described proteins have been described for purifyingimmunoglobulins, with a Protein A being most commonly described inantibody purification. Very generally, Protein A, Protein G, Protein L,or Protein A/G are used in affinity chromatography to bind targetantibodies from samples. The antibodies (bound to Protein A, G, L, orA/G) can then be separated from most other components of sample, andoptionally further purified using other purification steps.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, a polypeptide comprising a modified Protein G,Protein L, or Protein A/G that lacks antibody-binding activity isprovided. In some embodiments, the modified Protein G, Protein L, orProtein A/G comprises one or more modified amino acids whosemodification blocks antibody-binding activity. In some embodiments, themodified Protein G, Protein L, or Protein A/G comprises one or moremodified lysine amino acids. In some embodiments, the modified lysineamino acids are acetylated lysines.

In some embodiments, the modified Protein G, Protein L, or Protein A/Gcomprises one or more Fc fragment-comprising proteins cross-linked at ornear the binding sites on Protein G, Protein L, or Protein A/G, therebysterically blocking antibody-binding activity.

In some embodiments, the modified Protein G, Protein L, or Protein A/Gcomprises one or more amino acid changes compared to an antibody-bindingProtein G, Protein L, or Protein A/G, respectively, such that thepolypeptide lacks antibody-binding activity but retainscontaminant-binding activity.

In some embodiments, the modified Protein G, Protein L, or Protein A/Gcomprises sterically-blocked wildtype (or an antibody-binding variantof) Protein G, Protein L, or Protein A/G, respectively, such that thepolypeptide lacks antibody-binding activity but retainscontaminant-binding activity.

Also provided is a polypeptide comprising a modified Protein A thatlacks antibody-binding activity, wherein the modified Protein Acomprises one or more modified amino acids that block antibody-bindingactivity. In some embodiments, the modified Protein A comprises one ormore modified lysine amino acids. In some embodiments, the modifiedlysine amino acids are acetylated lysines.

Also provided is a solid support or support matrix linked to apolypeptide comprising a modified Protein A, Protein G, Protein L, orProtein A/G that lacks antibody-binding activity between 0-30° C. Insome embodiments, the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more modified amino acids that blockantibody-binding activity. In some embodiments, the modified Protein A,Protein G, Protein L, or Protein A/G comprises one or more modifiedlysine amino acids. In some embodiments, the modified lysine amino acidsare acetylated lysines.

In some embodiments, the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more Fc fragment-comprising proteinscross-linked to the Protein G, Protein L, or Protein A/G, therebyblocking antibody-binding activity.

In some embodiments, the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more amino acid changes compared towildtype (or an antibody-binding variant of) Protein G, Protein L, orProtein A/G, respectively, such that the polypeptide lacksantibody-binding activity but retains contaminant-binding activity.

In some embodiments, the support matrix is a bead, membrane, or fiber.

Also provided are reaction mixtures comprising an antibody-comprisingsample and (1) a modified Protein A, Protein G, Protein L, or ProteinA/G that lacks antibody-binding activity, e.g., as described above orelsewhere herein or (2) a modified Protein A that lacks antibody-bindingactivity, wherein the modified Protein A comprises one or more modifiedamino acids that block antibody-binding activity.

Also provided are methods of purifying antibodies from sample comprisingthe antibodies and contaminants. In some embodiments, the methodcomprises contacting the sample with a polypeptide comprising a modifiedProtein A, Protein G, Protein L, or Protein A/G that lacksantibody-binding activity under conditions in which at least somecontaminants bind to the polypeptide and the antibodies do not bind tothe polypeptide; separating a solution comprising the antibodies fromthe contaminants bound to the polypeptide; contacting the solutioncomprising the antibodies to an antibody-binding Protein A, Protein G,Protein L, or Protein A/G under conditions to form a complex of theantibodies and the antibody-binding Protein A, Protein G, Protein L, orProtein A/G, thereby generating the complex and unbound components;separating the unbound components from the complex; and eluting theantibodies from the complex, thereby generating purified antibodies.

In some embodiments, the polypeptide is linked to a support matrix. Insome embodiments, the support matrix is a bead, membrane, or fiber. Insome embodiments, the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more modified amino acids that blockantibody-binding activity. In some embodiments, the modified Protein A,Protein G, Protein L, or Protein A/G comprises one or more modifiedlysine amino acids. In some embodiments, the modified lysine amino acidsare acetylated lysines.

In some embodiments, the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more Fc fragment-comprising proteinscross-linked to the Protein A, Protein G, Protein L, or Protein A/G,thereby blocking antibody-binding activity.

In some embodiments, the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more amino acid changes compared to anantibody-binding Protein A, Protein G, Protein L, or Protein A/G,respectively, such that the polypeptide lacks antibody-binding activitybut retains contaminant-binding activity.

Also provided are methods of purifying antibodies from sample comprisingthe antibodies and contaminants, comprising, contacting the sample to anantibody-binding Protein A, Protein G, Protein L, or Protein A/G underconditions to form a complex of the antibodies and the active Protein A,Protein G, Protein L, or Protein A/G, thereby generating the complex andunbound components; separating the unbound components from the complex;eluting the antibodies from the complex, to form an eluate comprisingthe antibodies; contacting the eluate with a polypeptide comprising amodified Protein A, Protein G, Protein L, or Protein A/G that lacksantibody-binding activity under conditions in which at least somecontaminants bind to the polypeptide and the antibodies do not bind tothe polypeptide; and separating a solution comprising the antibodiesfrom the contaminants bound to the polypeptide; thereby generatingpurified antibodies.

In some embodiments, the polypeptide is linked to a support matrix. Insome embodiments, the support matrix is a bead, membrane, or fiber. Insome embodiments, the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more modified amino acids that blockantibody-binding activity. In some embodiments, the modified Protein A,Protein G, Protein L, or Protein A/G comprises one or more modifiedlysine amino acids. In some embodiments, the modified lysine amino acidsare acetylated lysines.

In some embodiments, the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more Fc fragment-comprising proteinscross-linked to the Protein A, Protein G, Protein L, or Protein A/G,thereby blocking antibody-binding activity.

In some embodiments, the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more amino acid changes compared towildtype or an antibody-binding variant of Protein A, Protein G, ProteinL, or Protein A/G, respectively, such that the polypeptide lacksantibody-binding activity but retains contaminant-binding activity.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

Provided are modified versions of proteins commonly used in antibodypurification as well as kits and methods for their use. Specifically,modified Protein A, Protein G, Protein L, and Protein A/G are providedthat lack antibody-binding activity. These polypeptides are useful inantibody purification in conjunction with native antibody-bindingactivity Protein A, Protein G, Protein L, and Protein A/G (i.e., havingantibody binding activity) by removing contaminants that bind to nativeProtein A, Protein G, Protein L, and Protein A/G and thus end up ascontaminants in antibody preparations.

Accordingly, in some embodiments, a method is provided of contacting anantibody sample with a modified Protein A, Protein G, Protein L, orProtein A/G in the course of purification, thereby removing contaminantsthat would have, or that have already, co-purified with the antibodies.Thus, in some embodiments, a protein sample is contacted with a nativeor an antibody-binding variant of Protein A, Protein G, Protein L, orProtein A/G under conditions to bind antibodies in the sample andallowing for purification of the antibodies from at least some othercomponents of the sample. The resulting purified antibody mixture cannevertheless subsequently contain contaminants that also bound to thenative or an antibody-binding variant of Protein A, Protein G, ProteinL, or Protein A/G. These contaminants can be removed by subsequently(immediately after or after intervening purification steps) contactingthe purified antibody mixture with a modified Protein A, Protein G,Protein L, or Protein A/G lacking antibody-binding activity. Thecontaminants that bound the native or an antibody-binding variant ofProtein A, Protein G, Protein L, or Protein A/G will bind to themodified Protein A, Protein G, Protein L, or Protein A/G whereas theantibodies in the mixture will not. Thus, the antibodies can beseparated from the bounds contaminants, resulting in a more pureantibody preparation.

Alternatively, in some embodiments, the modified Protein A, Protein G,Protein L, or Protein A/G can be contacted to a protein sample underconditions to allow any contaminants with affinity for native Protein A,Protein G, Protein L, or Protein A/G to bind to the modified Protein A,Protein G, Protein L, or Protein A/G, thereby removing suchcontaminants. The resulting protein mixture (reduced for Protein A,Protein G, Protein L, or Protein A/G-binding contaminants) can then besubsequently (immediately after or after intervening purification steps)contacted with native or an antibody-binding variant of Protein A,Protein G, Protein L, or Protein A/G, to bind and affinity purify theantibodies in the mixture, again resulting in a more pure antibodypreparation compared to an equivalent purification scheme lacking themodified Protein A, Protein G, Protein L, or Protein A/G step.

The methods may be combined with other purification methods to achievehigher levels of purification. Examples include, but are not limited to,other methods commonly used for purification of antibodies, such asother forms of affinity chromatography, anion exchange chromatography,cation exchange chromatography, hydrophobic interaction chromatography,immobilized metal affinity chromatography, and additional mixed modechromatography methods. Other options, include, but are not limited toprecipitation, crystallization, and/or liquid partitioning methods.

II. Modified Protein A, G, L or A/G

Any type of modified Protein A, G, L, or A/G polypeptide that lacksantibody-binding activity can be used in the methods described herein. Amodified Protein A, G, L, or A/G “polypeptide that lacksantibody-binding activity” refers to a modified Protein A, G, L, or A/Gthat has no more than 10% of a native antibody-binding (i.e., Fcdomain-binding) polypeptide activity, e.g., the modified Protein A, G,L, or A/G has less than 1/10, 1/100, or 1/1000 the binding activity ofthe native Protein A, G, L, or A/G. While it is appreciated that a“native” Protein A/G does not exist, for the purposes of thisdisclosure, “native Protein A/G” refers to a recombinant fusion proteinthat combines native IgG binding domains of both Protein A and ProteinG. Protein A/G contains four Fc binding domains from Protein A and twofrom Protein G as known in the art.

Various variant forms of Protein A, G, L and A/G are known that bindantibodies. One such is MabSelect SuRe™, which is a native protein Awith amino acid modifications designed to improve alkali stability. Asused herein, “wildtype”; and “native” are used synonymously to mean anaturally-occurring protein.

Examples of modifications that reduce or eliminate antibody-bindingactivity of Protein A, G, L, or A/G include, but are not limited to,amino acid modification, amino acid mutation, and/or blocking ofantibody-binding sites of Protein A, G, L, or A/G.

Modified Amino Acids

“Modified amino acids,” as used herein, refers to chemical modificationof naturally-encoded amino acids, resulting in reduction ofantibody-binding to the modified protein. A variety of particularnaturally-encoded amino acids, due to their particular chemicalstructure, are selectively reactive to specific chemicals, resulting inmodification of the particular amino acid type without significantmodification of other amino acids not of that type in the protein.

A number of examples of amino acid modification are known and can beapplied to eliminate antibody-binding activity of Protein A, G, L, orA/G. For example, in one embodiment, lysine residues in the protein aremodified by acetylation. This can be achieved, for example, bycontacting the protein with a sufficient amount of acetic anhydride. Inanother embodiment, arginine residues in the protein are citrullinated(deaminated). In yet other embodiments, glutamine and asparagine aredeamidated, converting the residues to glutamic acid and aspartic acid,respectfully. Yet other embodiments allow for eliminylation, i.e., theconversion to an alkene by beta-elimination of phosphothreonine andphosphoserine, or dehydration of threonine and serine. Other sorts ofchemical modification of amino acids (e.g., as known in the art) inProtein A, G, L, or A/G can also be used.

Modified amino acids that inhibit the antibody-binding activity ofProtein A, G, L, or A/G can also be formed in two or more steps. As anexample, in one embodiment, one type of amino acid is converted to asecond type of amino acid (e.g., arginine to lysine) which itself doesnot affect antibody-binding activity but then the second type of aminoacid is modified (e.g., acetylated) to reduce antibody-binding activityof the protein.

Mutations

In other embodiments, one or more amino acid mutations are introducedinto Protein A, G, L, or A/G to inhibit antibody-binding activity. Insome embodiments, the resulting mutation protein is at least 90, 95, or97% identical to the native or an antibody-binding variant of Protein A,G, L, or A/G. For example, in some embodiments, the modified proteincomprises at least one but no more than 2, 3, 4, 5, 6, 7, 8, or 9 aminoacid changes compared to native Protein A, G, L, or A/G. In someembodiments, the mutated Protein A, G, L, or A/G hastemperature-sensitive antibody-binding activity. Examples of suchproteins are described in, e.g., US Patent Publication No. 2013/0317172.In other embodiments, the mutated Protein A, G, L, or A/G does not haveantibody-binding activity at any temperature. For example, in someembodiments, the mutated Protein A, G, L, or A/G does not haveantibody-binding activity between at least between 0-30° C.

A variety of Protein A mutations have been described that lack Fc-domainbinding activity, albeit for other uses. See, e.g., US PatentPublication No. 2013/0171183. For instance, in some embodiments, atleast one of F5, Q9, Q10, S11, F13, Y14, L17, N28, I31, and/or K35 ofthe IgG Fc binding sub-domain of Protein A domain D is modified orsubstituted.

In addition to point mutations, one or more (e.g., 2, 3, 4, 5, 6, 7, 8,9, 10 or more, e.g., 10-20, 10-30 amino acids) amino acid deletions orinsertions can be made in Fc-binding regions of Protein A, G, L, or A/G,thereby blocking or eliminating antibody-binding activity.

Mutations can be introduced by random mutagenesis or by site-directedmutagenesis. Basic texts disclosing the general methods of mutagenesisand recombinant techniques include Sambrook and Russell, MolecularCloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994-1999).

Blocked Protein A

In yet another aspect, Protein A, G, L, or A/G can be treated with ablocking agent that blocks antibody-binding activity of Protein A, G, L,or A/G. For example, in some embodiments, Protein A, G, L, or A/G arecontacted with a Fc-domain containing proteins (e.g., a full-lengthantibody tetramer or fragment thereof) and the resulting complex iscross-linked. Exemplary cross-linking agents include, but are notlimited to, dimethyl suberimidate, the N-hydroxysuccinimide-estercrosslinker BS3, dicyclohexylcarbodiimide (which activates carboxylgroups to conjugate to primary amines), and formaldehyde. Cross-linkingprovides an advantage that the blocking agent will not disassociate fromthe Protein A, G, L, or A/G during the methods described herein forremoval of contaminants. However, cross-linking is not necessary inscenarios in which the complex of Protein A, G, L, or A/G and blockingagent are used under conditions that do not significantly disassociatethe complex.

Methods of Screening

As desired, further modified proteins with reduced antibody-bindingactivity can be screened for and selected. Thus, in some embodiments,Protein A, G, L, or A/G is contacted with a particular amino acidmodifying agent and/or blocking agent, and/or mutations are introduced(e.g., recombinantly) into Protein A, G, L, or A/G and the resultingproteins are screened for antibody-binding activity. Antibody-bindingactivity can be assayed by, for example, 1) using an immunoassay (e.g.,an enzyme-linked immunosorbent assay (ELISA)); 2) measuring bindingactivity directly via surface plasmon resonance; 3) Static or dynamiclight scattering; or other technique that measures protein-protein orantibody-protein binding. Proteins with reduced antibody-bindingactivity can then be selected. Using an ELISA, for example, reducedantibody binding would result in, e.g., a modified protein A thatproduced little or no signal from the ELISA assay (e.g., little or nofluorescence, visible light, radioactivity, etc., depending upon thereporter group).

III. Support Matrices

As noted above, in purifying target antibodies, it can be desirable tolink either native or an antibody-binding variant of Protein A, G, L, orA/G and/or modified Protein A, G, L, or A/G lacking antibody-bindingactivity to a solid support. This allows, for example, for separation ofcomponents of a mixture that binds to the native or modified Protein A,G, L, or A/G from other components of the mixture. As described above,in the case of native or an antibody-binding variant of Protein A, G, L,or A/G, the components binding the Protein A, G, L, or A/G are generallydesired antibodies whereas those components binding the modified ProteinA, G, L, or A/G are non-antibody components that otherwise occur ascontaminants in an antibody preparation.

A “solid support” refers to a material or group of materials having arigid or semi-rigid surface or surfaces. In some embodiments, the solidsupport takes the form of thin films or membranes, beads, bottles,dishes, fibers, woven fibers, shaped polymers, particles, andmicroparticles, including but not limited to, microspheres. A solidsupport can be formed, for example, from an inert solid support,including but not limited to, natural material, such as glass andcollagen, or synthetic material, such as acrylamide, cellulose,nitrocellulose, silicone rubber, polystyrene, polyethylene vinylacetate, polypropylene, polymethacrylate, polyethylene, polysilicates,polyethylene oxide, polycarbonates, teflon, fluorocarbons, nylon,polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters,polypropylfumarate, glycosaminoglycans, and polyamino acids. Frequently,some functional groups, e.g., carboxylic acid (—COOH), free amine(—NH2), and sulfhydryl (—SH) groups, naturally present on the surface ofa carrier can be used for peptide linkage. In case no such functionalgroup is naturally available, a desired functional group, such as acarboxylic acid group, or a moiety known to be a partner of a bindinginteraction (such as avidin that is capable of binding biotin) may beattached to such solid support. In some embodiments, the solid supportis a carboxylated latex or magnetic microsphere. Numerous methods areknown in the art for linking proteins to a solid surface. As oneexample, a linking agent (including but not limited toN-hydroxisulfosuccinimide (NHSS) optionally with1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)) canbe used to link a protein to a solid support.

In some embodiments, the solid support is a support matrix. A variety ofsupport matrices can be used. Generally, the support matrix will be ahydrophilic polymer that allows for linkage of the ligand, optionallyvia a spacer.

In some embodiments, the base matrix is hydrophilic and in the form of apolymer, e.g. a polymer that is insoluble and more or less swellable inwater. Suitable polymers include, but are not limited to polyhydroxypolymers, e.g. based on polysaccharides, such as agarose, dextran,cellulose, starch, pullulan, etc. and completely synthetic polymers,such as polyacrylic amide, polymethacrylic amide, poly(hydroxyalkylvinylethers), poly(hydroxyalkylacrylates) and polymethacrylates (e.g.polyglycidylmethacrylate), polyvinyl alcohols and polymers based onstyrenes and divinylbenzenes, and copolymers in which two or more of themonomers corresponding to the above-mentioned polymers are included.Polymers, which are soluble in water, may be derivatized to becomeinsoluble, e.g. by cross-linking and by coupling to an insoluble bodyvia adsorption or covalent binding. Hydrophilic groups can be introducedon hydrophobic polymers (e.g. on copolymers of monovinyl anddivinylbenzenes) by polymerisation of monomers exhibiting groups whichcan be converted to OH, or by hydrophilization of the final polymer,e.g. by adsorption of suitable compounds, such as hydrophilic polymers.In some embodiments, the support is an UNOsphere™ support, a polymerproduced from water-soluble hydrophilic monomers (Bio-Rad, Hercules,Calif.). Alternatively, the matrix is agarose (GE Sepharose or SterogeneSuperflow and Ultraflow).

IV. Kits

In some embodiments, the modified Protein A, G, L, or A/G that lacksantibody-binding activity is packaged in a kit. In some embodiments, thekits further comprise other reagents, for example other reagents asdescribed herein. For example, in some embodiments, the kit comprises atleast one container comprising modified Protein A, G, L, or A/G and asecond container comprising native or an antibody-binding variant ofProtein A, G, L, or A/G. Instructions can optionally be provided in orwith the kit.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A support matrix linked to a polypeptidecomprising a modified Staphylococcus aureus Protein A, Protein G,Protein L, or Protein A/G that lacks antibody-binding activity between0-30° C., wherein the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more acetylated lysine amino acids thatblock antibody-binding activity or wherein the modified Protein A,Protein G, Protein L, or Protein A/G comprises one or more Fefragment-comprising proteins cross-linked to the Protein G, Protein L,or Protein A/G, thereby blocking antibody-binding activity.
 2. Thesupport matrix of claim 1, wherein the modified Protein A, Protein G,Protein L, or Protein A/G comprises one or more acetylated lysine aminoacids that block antibody-binding activity.
 3. The support matrix ofclaim 1, wherein the modified Protein A, Protein G, Protein L, orProtein A/G comprises one or more Fc fragment-comprising proteinscross-linked to the Protein G, Protein L, or Protein A/G, therebyblocking antibody-binding activity.
 4. The support matrix of claim 1,wherein the support matrix is a bead, membrane, or fiber.